Background
Epigenetic regulation of ribosomal DNA (rDNA) locus has a pivotal role in orchestrating ribosome biogenesis. Human cells contain about 400 copies of the ribosomal RNA (rRNA) genes organized as tandem, head-to-tail repeats [
1,
2], which are located in the fibrillar centers and the dense fibrillar component of the nucleolus [
3]. Each unit is ~43 kb long and includes the 47S rRNA encoding sequence (~13 kb) and a non-transcribed intergenic spacer (~30 kb). In physiological conditions, around half of these copies is allelically inactivated through a combination of epigenetic mechanisms including late replication time [
4], specific repression factors [
5,
6] and methylation of rDNA promoter. rDNA promoter includes a core promoter region, extending from −50 to +20 in respect to the transcription starting site (TSS), and an upstream control element (UCE) at −200 in respect to TSS. In humans, but not in rodents, both the UCE and the core promoter are CpG rich regions, classifiable as CpG islands, which usually show a complex methylation pattern [
7,
8] that can affect rRNA expression [
9,
10].
Bisulfite sequencing of clonal rDNA promoters has been used to characterize rDNA methylation status in several pathological conditions. Hypermethylation of rDNA promoter was described in brain from Alzheimer’s disease [
11] and suicide subjects [
12], while methylation levels of 18S and 28S 5’ regions were decreased in white blood cells from systemic lupus erythematosus subjects [
13]. rDNA hypermethylation occurs during aging [
14], and accordingly accelerated methylation of ribosomal regions was shown in fibroblasts from subjects affected by Werner syndrome [
15]. The analysis of rDNA methylation in tumor samples appears to be in this context of extreme interest. Ribosome biogenesis is a limiting factor in sustaining the increased demand for protein synthesis, a prerequisite for cell growth and cell proliferation [
16,
17], and, as consequence, the rate of ribosome production is notably enhanced in cancer cells. rDNA promoter was found hypomethylated in respect to corresponding normal tissue in human hepatocellular carcinomas [
7] but not in prostate cancer [
18]. On the contrary, Yan and colleagues used methylation-sensitive Southern blotting to show increased rDNA methylation in patients with breast cancer compared to the normal control tissue; rDNA hypermethylation resulted also in association with specific tumor features such as the negativity of oestrogen receptors and poor tumor differentiation status [
19].
In this study we analyzed methylation levels of three different regions within rDNA genes (the promoter and 5’ regions of 18S and 28S sequences). In order to precisely define rDNA methylation profiles in breast cancer we used the MassARRAY EpiTYPER assay, a more sensitive and quantitative method compared to Southern blot and to clonal sequencing. Furthermore, we investigated a possible correlation between the methylation status of single CpG sites, ribosomal biogenesis and the available clinical and bio-pathological parameters in order to define its possible impact in the biological and clinical behavior of the tumors.
Discussion
DNA methylation is a key regulator of gene expression and of genome architecture, and defects in its regulation often occur in several human diseases, including cancer. As many other types of tumors, up to 50% of cases of breast cancer show hypomethylation of repetitive DNA sequences and transposable elements, which substantially contributes to genomic instability [
29]. Moreover, genome-wide studies on tumor tissues and breast cancer cell lines have reported aberrant hypermethylation of the CpG islands of several genes, including tumor suppressors [
30‐
33].
In this study, we specifically analyzed the methylation of rDNA genes in breast cancer tissues. Indeed, altered regulation of ribosome biogenesis is a common feature of many cancers, and it has been deeply investigated in breast tumors [
34]. In proliferating cancer cells, the rapidity of cell proliferation is strictly dependent on ribosome production [
24,
25,
35]. This is one of the major factors contributing to the growth rate of a tumor mass inside the host, which is one of the most important prognostic factors in oncology. In human carcinomas the association of nucleolar hypertrophy with bad prognoses is noteworthy and there is an increasing amount of data that suggests an active role of the nucleolus in tumorigenesis [
3,
36]. In line with this, ribosome synthesis has been also identified as a promising target for antineoplastic therapy [
37‐
44].
The methylation status of rDNA promoter, which is CpG-rich in human [
45‐
47], was investigated in breast cancer tissues respect to matched normal tissues. The methylation of two CpG-rich regions located at the 5’ of 18S and 28S sequences was considered too. Our data indicate an increased rDNA methylation in tumors compared to normal tissues. Although this finding is unexpected, as neoplastic transformation should sustain ribosome biogenesis and therefore rDNA hypomethylation, similar results have been previously described. Yan and colleagues showed increased rDNA methylation levels in breast cancer biopsies compared to normal control tissue and found that rDNA hypermethylation was associated with the oestrogen receptor negative and with moderately or poorly differentiated tumors [
19]. The technical approach employed in this work provided information about overall methylation status of rDNA, regardless to the epigenetic regulation of specific CpG sites [
19]. Our results confirmed rDNA hypermethylation in breast tumors using the MassARRAY EpiTYPER assay, a technique that allows to assess methylation levels with single base resolution and that is more sensitive and quantitative compared to Southern blot and to clonal-sequencing of bisulfite-treated DNA. This approach allowed us to deeply characterize DNA methylation profile of rDNA. As expected, in normal tissues the CpG sites in the promoter showed a strong correlation in their methylation levels. Moreover, also the methylation status of the CpG sites within the gene body (18S and 28S regions) resulted highly correlated, suggesting a tight control over the entire region in normal conditions. Correlation levels were slightly but significantly lower in tumor samples respect to normal controls, indicating that a loss in the epigenetic control, which is a common characteristic of cancer, occurs also in rDNA region.
Surprisingly, rDNA methylation of normal breast tissues showed substantial inter-individual variation, ranging from 20% to 40% depending on the CpG site. The biological basis of this strong variability is not clear, although it should be considered that ribosomal biogenesis, and potentially also rDNA methylation, is strongly affected by environmental factors, such as the intracellular energy status [
48].
Yan and coworkers showed that higher rDNA methylation levels in tumor breast tissues were correlated with ER-negativity and suggested that they could be predictive of the tumor propensity to hypermethylate ER promoter. We did not find any significant association between rDNA methylation and ER status, but it should be considered that in our cohort ER negative cases were only a minor part of the samples (11/68 compared to 27/58 in the work by Yan [
19]). On the contrary, significant association was found between methylation values of several sites of rDNA loci and NG and nucleolar size values. Although only some CpGs reached statistical significance, the entire locus showed the same trend in terms of DNA methylation variations, confirming a common regulation of the CpG sites within the region. The nucleolar size evaluation after its selective staining with silver is a well established method used in tumor pathology for tumor characterization, being nucleolar hypertrophy associated with bad prognosis. Together with nuclear polymorphism, the presence of prominent nucleoli is one of the parameters influencing NG classification. We observed that a subgroup of samples with NG = 3 but nucleolar size ≤ 5 μm
2 showed higher rDNA methylation levels, suggesting that in breast tumors the methylation status of rDNA loci can affect the rate of ribosome biogenesis and somehow counteract other adverse pathological conditions. Accordingly, we identified a subgroup of patients in which the presence of large nucleoli was associated to limited or absent rDNA hypermethylation of tumor tissue respect to matched normal control. In these tumors the lack of rDNA hypermethylation could represent an important factor to cope with the need for a particularly intense biosynthetic activity. In addition, this observation confirms an epigenetic regulation of ribosomal biogenesis in breast cancer and indicates that the rate of rDNA hypermethylation can significantly differ between patients. Importantly, samples showing small tumor-normal tissues differences had higher nucleolar size, indicating that not only the rDNA methylation level, but also the extent of rDNA hypermethylation in respect to normal tissue could represent a marker of breast cancer progression and, in principle, could be explored as a potential prognostic marker for this tumor type.
rDNA hypermethylation was described in other women’s cancers, including ovarian cancer [
49] and endometrial carcinoma [
50]. The mechanisms and the dynamics that lead to rDNA hypermethylation in these tumors are not clear, also because tumor progression should in theory sustain higher levels or ribosome biogenesis, and therefore rDNA hypomethylation in respect to normal tissue. In all the cases, higher levels of rDNA methylation were associated to better prognosis and longer disease-free and overall survival, suggesting that rDNA methylation could have a role in the biological and clinical behavior of the tumors. One intriguing scenario is that rDNA hypermethylation may be a defense response against tumor progression, but further analyses are needed to explore this issue. With respect to previous studies, where the relationship between rDNA methylation and ribosomal biogenesis was not considered, we demonstrated for the first time that rDNA methylation is associated to nucleolar size in breast cancer. Future studies should assess if rDNA methylation affects the rate of rRNA transcription and therefore the proliferative potential of tumor cells.
Acknowledgements
We thank Vilma Mantovani and Elena Marasco for their technical support during experimental procedure of DNA methylation analysis at CRBA (Applied Biomedical Research Center, S. Orsola-Malpighi Polyclinic, Bologna, Italy). This work was supported by grants from the Italian Association for Cancer Research (IG-11416) to L. Montanaro and from European Union’s Seventh Framework Programme (IDEAL project, 259679) to C. Franceschi.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
LM, PG, SS, CF, MGB and AP conceived the study and wrote the article. MGB and AP developed the methodology. MGB, AP, MP, DT, CG, CP carried out the experimentation, acquired the data and performed statistical analysis. All authors read and approved the final manuscript for publication.